The combined AFM manipulation and fluorescence imaging of single DNA molecules
A combined fluorescence microscope/AFM set-up was constructed, which enabled the real-time manipulation of single DNA molecules. Fluorescence images of these TO-PRO-3 intercalated strands could be taken, while they were stretched with an AFM tip on silanised or polylysine covered glass surfaces. Characteristic AFM force spectra of single DNA molecules were achieved and a statistical analysis of the rupture forces, plateau heights and rupture lengths was compiled. The wide-field fluorescence images indicated that the DNA underwent condensation on polylysine to form aggregated rods and globular structures. Due to the strong unspecific adhesion of the DNA to the polylysine surface, AFM tip manipulation frequently led to strand scission. In addition, it was possible to “write” nm-sized letters of fluorescent DNA by unraveling agglomerated strands from the tip onto the surface. In contrast, DNA strands on silane showed far less condensation. Extended single chains were bound to the surface by the termini or at specific sites along the double helix. These fixed and straightened strands could be overstretched laterally to ca. 1.6 times the original contour length. Chain rupture occurred at the tip position, but occasionally mid-strand rupture was also observed. An analysis of the chain fluorescence intensity for different stretching lengths revealed that the dyes remain intercalated up to the end of the DNA B-S overstretching transition.